PROJECT SUMMARY Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is the causative agent of AIDS malignancies like KS and primary effusion lymphomas (PEL). In recent years it became clear that pathogenic herpesviruses including EBV, KSHV, and MHV68 express numerous long non-coding RNAs (lncRNAs) many of which are in antisense orientation to protein coding transcripts. The function and structure of these RNAs is largely unknown. In addition, these viruses express microRNAs (miRNAs). While characterizing the KSHV miRNA targetome, we identified several hundred host cellular lncRNAs as putative miRNA targets. Furthermore, infection of endothelial cells with wtKSHV induced dramatic dys-regulation of host lncRNAs including the down-regulation of 533 lncRNAs. Of these 126 were rescued when cells were infected with a KSHV recombinant that lacked 10 of 12 KSHV miRNAs. Together these data strongly suggest that both KSHV encoded proteins and miRNAs contribute to dysregulation of host lncRNAs. Importantly, ten lncRNAs that are perturbed following KSHV infection, including HOTTIP, ANRIL, Meg3, and UCA1 are reported to be associated with human cancers. We demonstrate that up-regulation of UCA1 affects proliferation and migration of KSHV infected endothelial cells. Additionally, we provide experimental evidence that the anti-sense LANA transcript is bound by EZH2/PRC2 complexes and may contribute to the regulation of viral latency. These data suggest that viral lncRNAs are important regulators of viral gene expression and hence may be important for viral pathogenesis and/or tumorigenesis. To understand the role of lncRNAs in viral biology we propose to functionally study viral lncRNAs and to determine underlying mechanisms and phenotypical consequences of host lncRNA dysregulation in KS pathogenesis. Importantly, experimental findings observed in appropriate tissue culture models of lymphoid and endothelial origin, will be validated using clinical specimens from AIDS malignancies in collaboration with Dr. Chris Parsons. Moreover, this project will be performed in close collaboration with Projects 2 (EBV) and 3 (MHV68), with the goal of identifying pathways that are commonly regulated by cancer-associated gamma-herpesvirus lncRNAs. Identifying such common regulatory pathways may point to novel virus-specific therapeutic strategies. We note that to date several miRNA and lncRNA based therapeutics are in different stages of clinical development.